www.impactjournals.com/oncotarget/ Oncotarget, Vol. 6, No.

30

Effect of latent membrane protein 1 expression on overall survival in

Epstein-Barr virus-associated cancers: a literature-based meta-analysis
Yu-Pei Chen1,*, Wen-Na Zhang1,*, Lei Chen1,*, Ling-Long Tang1, Yan-Ping Mao1,
Wen-Fei Li1, Xu Liu1, Guan-Qun Zhou1, Ying Sun1, Tie-Bang Kang1, Mu-Sheng
Zeng1, Na Liu1, Jun Ma1
1
 un Yat-sen University Cancer Center; State Key Laboratory of Oncology in South China; Collaborative Innovation Center
S
for Cancer Medicine, Guangzhou, People’s Republic of China
*
These authors have contributed equally to this work
Correspondence to:
Na Liu, e-mail: liun1@sysucc.org.cn
Jun Ma, e-mail: majun2@mail.sysu.edu.cn
Keywords: LMP1, EBV, cancer, survival, meta-analysis
Received: May 20, 2015      Accepted: August 07, 2015      Published: August 18, 2015

ABSTRACT
Latent membrane protein 1 (LMP1) is identified as the main transforming
oncoprotein of Epstein-Barr virus (EBV). LMP1 is frequently expressed in a variety of
EBV-associated cancers, including nasopharyngeal carcinoma (NPC), non-Hodgkin
lymphoma (NHL), Hodgkin disease (HD), and gastric cancer (GC). However, due to
conflicting results, the prognostic value of LMP1 expression on clinical outcomes in
EBV-associated cancers remains unclear. We performed a meta-analysis on 32 studies
with a total of 3752 patients to explore the association between LMP1 expression
and overall survival (OS) in EBV-associated cancers. Overall, LMP1 expression was
significantly associated with poorer OS (hazard ratio, HR = 1.51, 95% confidence
interval, CI, 1.13–2.03), irrespective of cancer type. Further analyses showed that
LMP1 expression correlated with poorer OS in NPC (HR = 2.48, 95% CI, 1.77–3.47)
and NHL patients (HR = 1.83, 95% CI, 1.07–3.15), but not in HD patients (HR =
0.98, 95% CI, 0.60–1.62) or GC patients (HR = 0.70, 95% CI, 0.44–1.12). Subgroup
analyses indicated that the age and geographical factors seemed to have an effect
on the clinical outcomes of HD patients with positive LMP1 expression. In conclusion,
LMP1 expression can be used as a prognostic biomarker in NPC, NHL, and certain HD
patients. This data suggests that novel therapies targeting LMP1 may improve clinical
outcomes for EBV-associated cancer patients.

INTRODUCTION Expression of LMP1 in EBV-associated cancers

Epstein-Barr virus (EBV) is a ubiquitous
tumorigenic human herpes virus carried in more than
90% of adult populations worldwide [1]. EBV has been
implicated in a number of human malignancies of either
epithelial or lymphoid origin, including nasopharyngeal
carcinoma (NPC), lymphoma, and gastric cancer (GC)
[2]. Different patterns of latent EBV gene expression are
observed in these tumors, which can alter the phenotype
and cause oncogenic transformation of EBV-infected
cells. Of these gene products, latent membrane protein
1 (LMP1) from EBV has been identified as the main
transforming oncoprotein of EBV [3].
is associated with the regulation of proliferation,
immortalization, invasion, and angiogenesis of tumor cells
[1–4]. LMP1 is an integral membrane protein comprising
three domains: a short cytoplasmic N-terminus, six
transmembrane spanning regions, and a large cytoplasmic
C-terminal tail [4]. It activates the tumor necrosis factor
receptor (TNFR) signaling pathway through recruitment of
TNFR-associated factors and other adaptor proteins, and
stimulates or inhibits several other signaling pathways,
including NF-kB and PI3-K/Akt [3, 4]. Through these
signaling events, LMP1 has been shown to transform
rodent fibroblast cells, cause B lymphocyte immortalization
in vitro, and induce hyperplasia in transgenic mice [5]. In

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 addition, as LMP1 resembles CD40 in its functionality,
it can be partially used to substitute for CD40 in vivo, to
enhance B lymphocyte proliferation [6]. Therefore, LMP1
is attracting considerable attention as a potential prognostic
biomarker and novel therapeutic target.
Despite the clinical implication of LMP1 expression,
its prognostic value on clinical outcomes across different
EBV-associated cancers remains unclear. While some
studies indicate that LMP1 expression is positively
associated with cervical lymph node metastasis [7] and
is an unfavorable prognostic factor in NPC [8, 9], others
found no significant association [10, 11]. In addition,
LMP1 expression is suggested to be an unfavorable
prognostic factor for non-Hodgkin lymphoma (NHL)
patients but has no effect on the overall survival (OS) of
Hodgkin disease (HD) patients [12–16]. Other studies
indicate that certain epidemiologic factors (e.g., age,
geographical factors, socioeconomic status, and so on)
might influence the prognostic impact of LMP1 expression
in lymphomas [12–16].
Due to these conflicting results, a comprehensive
analysis of the prognostic effects of LMP1 is warranted.
We conducted a meta-analysis of the literature to explore
the association between LMP1 expression and OS among
patients with different types of EBV-associated cancers.
An improved understanding of this issue will enhance
rational development of more targeted EBV-associated
cancer therapy, which has important public health and
clinical implications.
RESULTS
Eligible studies
A total of 793 citations were identified after our
initial search. After the selection procedure, 32 studies
that met our inclusion criteria were included in this
meta-analysis [8–11, 13–40]. Figure 1 summarizes the
flow chart of study selection. Of these 32 studies, eight
(25%) evaluated NPC [8–11, 17–20], 23 (72%) evaluated
lymphoma [13–16, 21–39], and one (3%) evaluated GC
[40]. Of the 23 studies evaluating lymphoma, nine (39%)
focused on NHL [13, 21–28], while the remaining 14
(61%) focused on HD [14–16, 29–39]. Three studies
were prospectively conducted [14, 36, 39], and the
remaining studies used retrospective cohort designs. The
characteristics of all included studies are presented in
Table 1.
A total of 3752 patients were analyzed for LMP1
status and its relationship to disease prognosis, of which
1464 (39%) were classified as LMP1 positive. In the
32 studies, 30 (94%) investigations detected the LMP1
expression by immunohistochemistry (IHC), one (3%)
used the polymerase chain reaction (PCR), and one (3%)
used in situ hybridization (ISH). It should be noted that
the primary detection method in four studies [29, 30, 37,
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40] was ISH detecting EBV encoded nuclear RNA-1
(EBER-1) and the positive/negative cases in these studies
were positive/negative for EBER-1 expression. In these
EBER-1 positive cases, the positive rates for LMP1
expression were 69% [29], 90% [30], 65% [37], and 93%
[40]. Geographically, 10 (31%) studies were conducted in
Europe and North America, 20 (62%) in Asia, one (3%) in
South America, and one (3%) in South Africa. The quality
of the included studies, as assessed by the Newcastle-
Ottawa Scale (NOS), ranged from five to eight stars, with
22 (69%) studies of high quality, and 10 (31%) of low
quality (Table 2).
Meta-analysis of the effect of LMP1 expression
and overall survival in EBV-associated cancers
The positive expression of LMP1 was statistically
associated with a poorer OS (hazard ratio, HR = 1.51; 95%
confidence interval, CI, 1.13–2.03; Figure 2) when including
all 32 studies; however, significant heterogeneity was
detected (I2 = 70%; P < 0.001). In NPC patients, the pooled
random-effects model showed a significantly poorer OS with
positive expression of LMP1 (HR = 2.48; 95% CI, 1.77–
3.47; Figure 2); no significant heterogeneity was observed
(I2 = 22%; P = 0.254). The estimated HR was 2.36 (95% CI,
1.77–3.15) using a fixed effects model. In NHL patients, a
significant association between LMP1 expression and poorer
OS was also observed (HR = 1.83; 95% CI, 1.07–3.15),
while no significant association was found in HD patients
(HR = 0.98; 95% CI, 0.60–1.62). However, significant
heterogeneity was found in studies on NHL (I2 = 61%;
P = 0.008) and HD patients (I2 = 72%; P < 0.001; Figure 2).
Only one study was included for GC, which showed that the
expression of LMP1 had no significant correlation with OS
(HR = 0.70; 95% CI, 0.44–1.12; Figure 2).
Heterogeneity, sensitivity analyses, and
publication bias
Significant heterogeneity was exhibited in studies
on NHL and HD patients. Of the nine studies evaluating
NHL, the study by Kanemitsu et al. [13] was a notable
outlier in that it indicated a significantly more favorable
prognosis in LMP1 positive patients (Figure 2). After
excluding this study, no significant heterogeneity existed
(I2 = 19%; P = 0.282), and the combined HR based on the
fixed effects model of the remaining eight studies was 2.25
(95% CI, 1.61–3.13).
Figure 3 shows the results of the subgroup analyses
for HD patients. The prognostic effects were similar
between the five predefined subgroups according to
size of study, cutoff value, primary detection methods,
median/mean follow-up time, and NOS scores. However,
the prognostic effects based on median/mean age and
geographical area appeared discordant. The combined
HR was 0.69 (95% CI, 0.34–1.43) for studies with a
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 Figure 1: Flow chart showing the selection of the 32 studies included in the meta-analysis. LMP1, latent membrane protein
1; OS, overall survival.
median/mean age <40 years, while a significantly poorer
OS was associated with LMP1 expression in studies
with a median/mean age ≥40 years (HR = 1.82; 95% CI,
1.08–3.09). No significant association between LMP1
expression and survival was found in patients from
Europe and North America (HR = 1.42; 95% CI, 0.90–
2.23), while a significantly better OS was associated with
LMP1 expression in patients from other areas, such as
Asia, South Africa and South America (HR = 0.24; 95%
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CI, 0.11–0.52). This may partly explain the substantial
heterogeneity observed when examining LMP1 expression
as a prognostic factor in HD patients.
Funnel plots with the Begg test and Egger test are
shown in Figure 4. With all 32 included studies, visual
inspection of the Begg and Egger funnel plots did not identify
substantial asymmetry (P = 0.446 using the Begg test, and
P = 0.893 using the Egger test), indicating that there was no
evidence of publication bias detected in this study (Figure 4).
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 Table 1: Characteristics of the 32 studies included in the meta-analysis
First author, Country Period Histology Detection Cutoff value No. of Median/ Median/mean Quality HR (95%
year method for detection subjects mean age follow-up time score CI) for
(LMP1+/ (range) (months) overall
LMP1−) survival
NPC
1992– 224 2.06
Chen, 2010 China NPC IHC IRS, ≥4 46 (14–86) NA 8
2002 (141/83) (1.16–3.64)
Hariwiyanto, 5.56 (1.87–
Indonesia NA NPC IHC H-score, >7 56 (27/29) (11–70)* NA 7
2010 16.50)
1998– Percentage, Median 1.26 (0.69
Kitagawa, 2013 Japan NPC IHC 74 (35/39) 45.24 7
2009 ≥10% >50 –2.28)
Percentage
1999– 56.2 2.73 (0.91
Li, 2009 China NPC IHC x staining 57 (24/33) 36 8
2003 (22–72) –8.17)
intensity, ≥1
Un
1979– Positive: 2.82 (0.88
Sarac, 2001 Turkey differentiated IHC 35 (10/25) 35 (5–71) 66 7
1993 detectable –8.98)
NPC
2001– Percentage, 4.72 (1.45–
Song, 2007 China NPC IHC 50 (25/25) 50.24* NA 8
2003 ≥10% 15.33)
1999– Nonkeratin Percentage, 3.17
Wang, 2008 China IHC 60 (24/36) 53 (18–79) 36–74 8
2003 NPC ≥25% (1.37–7.31)
Un Percentage
1990– 2.80
Zhu, 2004 China differentiated IHC x staining 60 (39/21) 38 (13–65) 56 8
1991 (1.30–6.04)
NPC intensity, ≥1
NHL
1994– Percentage, 45.4 2.59
Cao, 2008 China ENKL IHC 58 (47/11) 84 7
2000 ≥ 10% (10–78) (1.01–6.67)
1980– 1.68 (0.80
Hirose, 2006 Japan PTCL IHC NA 43 (14/29) 63 (17–86) 14 6
2004 –3.54)
1990– 52.5 7.02 (1.91–
Ishii, 2007 Japan ENKL PCR >40 copies/ml 20 (13/7) 34 7
2003 (28–71) 25.73)
Kanemitsu, 1996– 0.24 (0.07
Japan ENKL IHC NA 30 (22/8) 62 (27–85) 26.7 6
2012 2010 –0.80)
1983– 0.85 (0.19
Kuze, 1996 Japan BCL IHC NA 17 (6/11) 60 (35–82) 12 5
1995 –3.82)
138 51.6 3.49
Paydas, 2008 Turkey NA NHL IHC NA NA 5
(20/118) (16–82) (1.68–7.25)
Percentage
1995– 1.73 (0.86
Xu, 2009 China ENKL IHC x staining 62 (30/32) 41 (13–79) NA 7
2005 –3.46)
intensity, ≥1
mRNA
1974– 3.80 (0.92–
Yamamoto,1999 Japan TCL ISH positive in 25 (15/10) NA >36 7
1994 15.80)
tumor cells
2000– Positive: 1.28 (0.40
Zhao, 2005 China ENKL IHC 36 (6/30) 40 (16–71) 17.7 7
2004 detectable –4.02)
HD
1988– 3.00
Clarke, 2001 USA HL IHC NA 78 (51/27)†‡ (45–79)* 73 6
1994 (1.50–6.40)
Germany 1990– 842 13.7 3.00
Claviez, 2005 HL IHC NA 58.5 7
& Austria 2001 (263/579) (2.2–20.2) (1.22–7.39)
1991– Percentage, 0.60
Dinand, 2009 India cHL IHC 122 (113/9) 8 (2–14) 48 7
2004 ≥25% (0.10–4.90)

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 First author, Country Period Histology Detection Cutoff value No. of Median/ Median/mean Quality HR (95%
year method for detection subjects mean age follow-up time score CI) for
(LMP1+/ (range) (months) overall
LMP1−) survival
1985– 2.06
Enblad, 1999 Sweden HL IHC NA 117 (32/85) 45 (11–87) 130 7
1988 (0.71–6.00)
South 0.08
Engel, 2000 NA HL IHC NA 36 (24/12)‡ 8 (3–14) 4–150 7
Africa (0.02–0.45)
Glavina- 1980– 0.98
Croatia HL IHC NA 100 (26/74) 40 (13–84) NA 7
Durdov, 2001 1990 (0.42–2.32)
USA&
1984– Positive: 303 1.11
Herling, 2003 Italy & cHL IHC 30* 65 7
2000 detectable (61/242) (0.50–2.45)
Greece
2.13
Keresztes, 2005 Hungary NA HL IHC NA 109 (47/62) 31 (3–74) 83 6
(0.74–6.15)
Krugmann, 1974– 37.6 0.96
Austria cHL IHC NA 119 (31/88) 122 7
2003 1999 (14–83) (0.39–2.33)
Positive: 37.2 0.39
Morente, 1997 Spain NA HL IHC 140 (72/68) 65 8
detectable (5–83) (0.17–0.92)
1992– 161 0.71
Murray, 1999 UK HL IHC NA 33 (22–49) 86 6
1996 (41/120) (0.32–1.57)
1984– Percentage, 0.26
Naresh, 2000 India cHL IHC 110 (86/24)‡ 22 (4–61) 57 6
1988 ≥10% (0.08–0.88)
1994– 0.36 (0.08
Quijano, 2004 Colombia HL IHC NA 57 (32/25) (3–83)* 23.8 6
1998 -1.60)
1991– 3.12
Stark, 2002 UK HL IHC NA 70 (24/46) (60–91)* 62.5 8
1998 (1.36–7.11)
GC
1995– Percentage, 343 0.70
Lee, 2004 Korea GC IHC 55* 54 6
1996 ≥10% (63/280)‡ (0.44–1.13)

Abbreviations: cHL, classical Hodgkin lymphoma; CI, confidence interval; ENKL, extranodal NK/T-cell lymphoma,
nasal type; GC, gastric cancer; HD, Hodgkin disease; HR, hazard ratio; H-score, histochemistry score; IHC,
immunohistochemistry; IRS, immunoreactive score; ISH, in situ hybridization; LMP1, latent membrane protein 1; NA,
not available; NHL, non-Hodgkin lymphoma; NPC, nasopharyngeal carcinoma; PCR, polymerase chain reaction; PTCL,
peripheral T-cell lymphomas; TCL, T-cell lymphoma.
*
Median/mean age or range is not available.
†
The survival data was only available for patients aged older than 45 years.
‡
The positive/negative cases in these studies were positive/negative for Epstein-Barr virus encoded nuclear RNA-1 (EBER-1)
as detected by in situ hybridization. The positive rates of LMP1 detected by IHC were 69%, 90%, 65%, and 93% in the EBER-1
positive cases in the studies by Clarke, Engel, Naresh, and Lee, respectively.

DISCUSSION Instead, age and geographical factors seemed to affect

LMP1 has been implicated in the etiology of various
EBV-associated cancers. However, the direction and
magnitude of the prognostic effect of LMP1 expression,
and whether the outcome is consistent among different
patient subgroups, remains unresolved. Here we conducted
a meta-analysis of 3752 patients included in 32 studies,
and found that EBV-associated cancer patients with
positive expression of LMP1 had significantly poorer
survival than those with negative expression. Subgroup
analyses showed that LMP1 expression was a significant
unfavorable biomarker in NPC and NHL, but it had no
significant effect on survival of HD and GC patients.
the clinical outcomes of HD patients with positive LMP1
expression.
NPC is particularly common in south China,
reaching a peak incidence of 20–50 per 100 000 males,
and shows the most consistent worldwide association
with EBV [3, 41]. Our meta-analysis found that LMP1
expression was a strong risk factor for the prognosis
of NPC; the risk of death was 2.48 fold higher in NPC
patients with positive expression of LMP1. This was the
highest risk of death among all EBV-associated cancers
studied (i.e., NPC, NHL, HD, and GC).
There are a few possible underlying mechanisms
involved in the association between LMP1 expression and

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 Table 2: Quality assessment of eligible studies using the Newcastle-Ottawa Scale (NOS)
First author, Represen Selection Ascer Demonstration that Comparability Assessment Follow- Adequacy Total NOS
year tativeness of non tainment outcome was not based on the of outcome up long of follow- score (stars)
of exposed exposed of present at start of design or enough for up of
cohort cohort exposure study analysis outcomes cohorts
to occur
NPC

Chen, 2010 1 1 1 1 2 1 1 0 8
Hariwiyanto,
1 1 1 1 2 1 0 0 7
2010
Kitagawa, 2013 1 1 1 1 1 1 1 0 7
Li, 2009 1 1 1 1 2 1 1 0 8
Sarac, 2001 1 1 1 1 0 1 1 1 7
Song, 2007 1 1 1 1 1 1 1 1 8
Wang, 2008 1 1 1 1 1 1 1 1 8
Zhu, 2004 1 1 1 1 1 1 1 1 8
NHL

Cao, 2008 1 1 1 1 0 1 1 1 7
Hirose, 2006 1 1 1 1 1 1 0 0 6
Ishii, 2007 1 1 1 1 1 1 0 1 7
Kanemitsu,
1 1 1 1 1 1 0 0 6
2012
Kuze, 1996 1 1 1 1 0 1 0 0 5
Paydas, 2008 1 1 1 1 0 1 0 0 5
Xu, 2009 1 1 1 1 0 1 1 1 7
Yamamoto,1999 1 1 1 1 0 1 1 1 7
Zhao, 2005 1 1 1 1 0 1 1 1 7
HL
Clarke, 2001 1 1 1 1 0 1 1 0 6
Claviez, 2005 1 1 1 1 0 1 1 1 7
Dinand, 2009 1 1 1 1 0 1 1 1 7
Enblad, 1999 1 1 1 1 0 1 1 1 7
Engel, 2000 1 1 1 1 1 1 0 1 7
Glavina-
1 1 1 1 1 1 0 1 7
Durdov, 2001
Herling, 2003 1 1 1 1 1 1 1 0 7
Keresztes, 2005 1 1 1 1 0 1 1 0 6
Krugmann,
1 1 1 1 0 1 1 1 7
2003
Morente, 1997 1 1 1 1 1 1 1 1 8
Murray, 1999 1 1 1 1 1 1 0 0 6
Naresh, 2000 1 1 1 1 0 1 1 0 6
Quijano, 2004 1 1 1 1 1 1 0 0 6
Stark, 2002 1 1 1 1 1 1 1 1 8
GC
Lee, 2004 1 1 1 1 0 1 1 0 6

Abbreviations: HL, Hodgkin lymphoma; NHL, non-Hodgkin lymphoma; NPC, nasopharyngeal carcinoma; GC, gastric cancer.
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 Figure 2: Forest plot showing association of latent membrane protein 1 (LMP1) expression and overall survival
(OS). Pooled estimates of hazard ratio (HR) are based on random effects meta-analysis. Horizontal line represents 95% confidence interval
(CI). HD, Hodgkin disease; NHL, non-Hodgkin lymphoma; NPC, nasopharyngeal carcinoma; GC, gastric cancer.

poorer OS in NPC patients. LMP1 contributes to invasion
and metastasis by modulating cell-matrix interactions
through induction of matrix metalloproteinases (MMPs),
and downregulation of various metastasis suppressors [4, 8].
In addition, LMP1 modulates key tumor suppressor genes
and micro-RNAs, thereby imparting resistance to apoptosis
[4, 8]. Finally, LMP1 may trigger overall tumor cell growth,
motility and invasiveness through the activation of NF-kB,
JNK/p38-SAPK, PI3-K/Akt, ERK-MAPK and JAK/STAT
pathways [3, 4].
The incidence of lymphoma in the Chinese
population is approximately six cases per 100 000 people
[42]. We found that LMP1 expression was significantly
associated with poorer survival in NHL patients, possibly

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 Figure 3: Subgroup analyses showing association of latent membrane protein 1 (LMP1) expression and overall survival
(OS) according to various factors in Hodgkin disease (HD) patients. Median/mean age data was not available for the study by
Quijano et al. and median/mean follow-up (months) was not available for the studies by Engel et al. and Glavina-Durdov et al.. Pooled
estimates of hazard ratio (HR) are based on random effects meta-analysis. Horizontal line represents 95% confidence interval (CI). IHC,
immunohistochemistry; ISH, in situ hybridization.

due to similar mechanisms discussed above for NPC.
Moreover, LMP1 has the ability to immortalize resting
B lymphocytes and transform them into permanent,
latently infected lymphoblastoid cell lines, which plays
an important role in the etiology and the progression of
the disease [3, 5, 21, 25]. We observed heterogeneity in
our meta-analysis of NHL patients due to the study by
Kanemitsu et al., which unlike other studies, showed
improved OS was associated with LMP1 expression [13].
The results may have differed in this study because of
the relatively small sample size, regional distribution, or
different LMP1 variants used by Kanemitsu et al. [13].
Unlike NPC and NHL, we found no significant
association between LMP1 expression and survival in HD
patients. As a unique type of lymphoma, EBV-associated
HD has an unbalanced distribution: a relatively higher
proportion of EBV-positive cases are found among
children and elderly patients, whereas young adults are
usually EBV-negative. Additionally, the proportion of
EBV-positive cases in Europe and North America varies
from 27% to 51%, but the highest frequencies (70% to
100%) of EBV-positive cases are found in developing
countries (e.g., India, South Africa) [14, 43, 44]. Therefore,
the prognostic effect of LMP1 expression on HD may be
affected by a variety of factors, including the patients’
age and geographical location. This may explain why
we found no significant association between LMP1 and
survival in the entire HD patient population in our study
compared with NPC or NHL patients. In fact, our subgroup
analyses showed that age and geographical region do
affect the prognosis of HD patients with LMP1 expression.
Particularly, LMP1 expression was significantly associated
with poorer OS in studies where the patients’ median/
mean age was ≥40 years (HR = 1.82) but had a tendency
to improve OS in studies where patients’ median/mean
age was <40 years (HR = 0.69). In studies from Europe

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 Figure 4: Funnel plots with A. Begg test and B. Egger test showing association of latent membrane protein 1 (LMP1)
expression and overall survival (OS) in all 32 included studies. Visual inspection of the Begg and Egger funnel plots did not
identify substantial asymmetry. HR, hazard ratio; S.E., standard error.

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 and North America, LMP1 expression had a tendency to
worsen OS (HR = 1.42), while it improved OS in patients
from other geographical regions (HR = 0.24). There
are a few reasons to explain why age and geographical
regions have an effect on the prognosis of HD patients
with LMP1 expression. In HD, LMP1 expression may
have reciprocal effects on the expressing cells. As LMP1
has antigenicity, one study found that cytotoxic T-cells
infiltrated into the lesion in LMP1-positive HD cases and
this was associated with tumor regression [45]. Therefore,
although LMP1 may induce a malignant phenotype, it may
also elicit a more effective immune response, resulting in
the favorable outcomes in some HD cases [13, 35]. It is
possible that there is a beneficial LMP1-induced immune
response within younger people, while in older patients,
with relatively lower immunocompetence, this response
may be less effective [31, 39]. Similarly, in developing
countries, children have a relatively high risk of being
exposed to a wide spectrum of infectious agents from an
early age, so HD patients from these countries may have
a more efficient immune response induced by LMP1
expression [30, 37]. These factors may partly explain
the different prognostic effects of LMP1 in the different
age and geography groups in our study, although further
studies are needed to support these findings.
Finally, while EBV is found in ~10% of typical
gastric adenocarcinomas, which accounts for up to 75 000
new cases per year [3]. There has been considerable
controversy with respect to the prognostic effect of LMP1
on survival of GC patients [46]; Unfortunately, there
are simply too few studies on GC patients to draw any
meaningful conclusions. As only one study with a total of
63 LMP1-postive GC patients was included in our meta-
analysis, the precise role of LMP1 in the prognosis of GC
requires further investigation.
Our study demonstrates that LMP1 can help identify
patients with EBV-associated cancers who are at a high
risk for a poor clinical outcome. In addition to serving as
a prognostic biomarker, our results suggest development
of anti-LMP1 drugs could be a novel therapeutic strategy
for NPC, NHL, and certain HD patients (i.e., depending
on the patient’s age and geographical location). New
immunotherapy approaches, for example, administering
anti-LMP1 cytotoxic T cells to target the malignant cell
population that express LMP1, have been gaining interest
[36, 47, 48]. In addition, inhibition of LMP1 expression
by siRNA may be a good prospect; LPM1 siRNA has been
shown to induce cell cycle arrest and enhance sensitivity
to cisplatin in an EBV-positive NPC derived cell line [49].
The limitations of this meta-analysis should be
acknowledged. First, this is a literature-based analysis and
may have resulted in publication bias as predominantly
positive results were reported. However, no evidence
of publication bias was detected from the funnel plots.
Second, we extracted all information from published data,
rather than investigating individual patient data (IPD).
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This limited our ability to gain sufficient information
for further analyses, and restrain us to examine the
sources of heterogeneity using subgroup analyses in
HD patients. Third, as we only focus on the prognostic
effects of LMP1 in this study, and most of the eligible
studies did not provide data regarding progression-free
survival or disease-free survival. Therefore, we only used
OS data for this meta-analysis. Although OS is currently
the gold standard primary endpoint for survival analysis,
further studies with IPD are still needed to evaluate the
relationship between LMP1 and staging, and with other
survival endpoints, including progression-free survival,
disease-free survival, among others.
In conclusion, our meta-analysis shows that LMP1
expression can be used as a prognostic biomarker in
NPC, NHL, and certain HD patients. This knowledge
may assist in improving poor patient prognosis, and in
designing novel therapeutic targets for EBV-associated
cancers. Still, it should be noted that the development of
cancer is multifactorial; various factors (e.g., smoking,
diet, and environmental factors) may have an impact on
the prognostic effect of LMP1 expression. Therefore,
future studies with a large prospective design are required
to evaluate multiple factors simultaneously, and confirm
the clinical significance of LMP1 expression in EBV-
associated cancers.
MATERIALS AND METHODS
Literature search strategy and study selection
This meta-analysis was conducted in accordance
with the Preferred Reporting Items for Systematic Reviews
and Meta-Analyses (PRISMA) statement [50]. Relevant
studies published before May 2015 were identified through
searching the following electronic databases: PubMed,
Embase, Cochrane Library, Chinese National Knowledge
Infrastructure, and Wanfang Database. The following
search terms were used: 1) LMP1, latent membrane
protein 1; 2) EBV, Epstein-Barr virus; 3) cancer, tumor,
neoplasm, carcinoma; 4) survival, prognosis, prognostic
factor. The analysis was supplemented by a manual search
of reference lists of relevant review articles, and relevant
books, and by correspondence with study investigators.
The research work was examined without language limits.
All studies identified initially were screened by titles
and/or abstracts before full text of the studies that satisfied
our inclusion criteria were retrieved. The selection criteria
for eligible studies in this study were: 1) the exposure
of interest were cancer and LMP1; 2) the outcome of
interests were overall survival (OS) associated with
LMP1 status; 3) hazard ratio (HR) and the corresponding
95% confidence interval (CI) was either reported directly
or there was sufficient data provided in the studies to
calculate these values. If the studies based on the same
patient populations were reported in different publications,
29320 Oncotarget
 only the most completed or the latest publication was
included in this meta-analysis. Two researchers (YPC and
WNZ) assessed the study eligibility independently, and
any discrepancies were resolved by consensus.
Data collection and extraction
Two independent investigators (YPC and WNZ)
reviewed the publications and extracted the data. The
following details were extracted: lead author, year
of publication, country of origin, inclusion period,
cancer type, study design, detection method, cutoff
value detection, total number of patients, LMP1 status,
median/mean age, median/mean follow-up duration, and
assessments of outcomes (HR and the corresponding
95% CI of OS). If HR was not displayed directly, it was
estimated according to the methods described by Parmar
et al. [51].
Quality assessment
Two independent reviewers (YPC and WNZ) used
the Newcastle-Ottawa Scale (NOS) to assess the quality
of all included studies [52]. This scale is an eight-item
instrument to evaluate a study in three domains: selection
of participants, study comparability, and the ascertainment
of outcomes of interest. It uses the awarding of points,
or “stars”, to compare study quality in a quantitative
manner, with a maximum of nine stars. Studies with 7–8,
5–6, 4 and 0–3 stars were identified as very good, good,
satisfactory or unsatisfactory in quality, respectively [53].
In this meta-analysis, all included studies were identified
as very good or good in quality. To further minimize
potential bias, we judged studies that received a score of
≥7 stars to be of high quality, and those that scored <7
stars to be of low quality, and performed the subgroup
analysis accordingly.
Statistical analysis
HRs for OS with 95% CIs according to the
expression status of LMP1 were pooled. The OS was
defined as the time from diagnosis to death from any
cause or to the date of the last follow-up. HR was the only
summary statistic calculated for this analysis, as it allows
for censoring and considers the time to an event taking
into account the whole survival time. A combined HR > 1
reflected a shorter survival for LMP1 positive patients.
The significance of the pooled HR was determined by the
Z test, and a P-value < 0.05 was considered statistically
significant.
Heterogeneity across studies was evaluated by a
χ2-based test; the I2 statistic, a quantitative measure of
inconsistency across studies [54], was also calculated.
Statistically significant heterogeneity was defined as a χ2
P-value < 0.10 or an I2 statistic >50%. The combinations
www.impactjournals.com/oncotarget
of the estimated risks were computed by fixed-effect
models and random-effect models. If the results were
without heterogeneity, the fixed-effects model was used; if
the results showed heterogeneity, the summary estimation
was based on the Dersimonian and Laird random-effects
model [55]. Because characteristics of populations and
other confounding factors might not be consistent between
studies, we conducted further subgroup analysis to explore
the sources of heterogeneity.
Potential publication bias was assessed by visual
inspection of the Begg and Egger funnel plots; the Begg
and Egger tests were also performed at the P-value < 0.10
level of significance [56, 57]. All analyses were conducted
using STATA version 12.0 (Stata Corporation, College
Station, TX). All statistical tests were two-sided.
CONFLICTS OF INTEREST
The authors have declared no conflicts of interest.
GRANT SUPPORT
This work was supported by grants from the
Health & Medical Collaborative Innovation Project of
Guangzhou City, China (201400000001), the Science
and Technology Project of Guangzhou City, China
(14570006), the National Science & Technology Pillar
Program during the Twelfth Five-year Plan Period
(2014BAI09B10), the Planned Science and Technology
Project of Guangdong Province (2013B020400004),
the Key Laboratory Construction Project of Guangzhou
City, China (121800085), the National Natural Science
Foundation of China (81302366) and the Medical Science
and Technology Research Foundation of Guangdong
Province (B2013148).
REFERENCES
1. Macsween KF, Crawford DH. Epstein-Barr virus-recent
advances. The Lancet Infectious diseases. 2003; 3:131–140.
2. Young LS, Murray PG. Epstein-Barr virus and onco-
genesis: from latent genes to tumours. Oncogene. 2003;
22:5108–5121.
3. Young LS, Rickinson AB. Epstein-Barr virus: 40 years on.
Nat Rev Cancer. 2004; 4:757–768.
4. Dawson CW, Port RJ, Young LS. The role of the EBV-
encoded latent membrane proteins LMP1 and LMP2 in the
pathogenesis of nasopharyngeal carcinoma (NPC). Semin
Cancer Biol. 2012; 22:144–153.
5. Li HP, Chang YS. Epstein-Barr virus latent membrane
protein 1: structure and functions. J Biomed Sci. 2003;
10:490–504.
6. Uchida J, Yasui T, Takaoka-Shichijo Y, Muraoka M,
Kulwichit W, Raab-Traub N, Kikutani H. Mimicry of
29321 Oncotarget
 CD40 signals by Epstein-Barr virus LMP1 in B lymphocyte
responses. Science. 1999; 286:300–303.
7. Zhao Y, Wang Y, Zeng S, Hu X. LMP1 expression is posi-
tively associated with metastasis of nasopharyngeal carci-
noma: evidence from a meta-analysis. J Clin Pathol. 2012;
65:41–45.
8. Chen J, Hu CF, Hou JH, Shao Q, Yan LX, Zhu XF, Zeng YX,
Shao JY. Epstein-Barr virus encoded latent membrane pro-
tein 1 regulates mTOR signaling pathway genes which pre-
dict poor prognosis of nasopharyngeal carcinoma. Journal
of translational medicine. 2010; 8:30.
9. Hariwiyanto B, Sastrowiyoto S, Mubarika S, Salugu M.
LMP1 and LMP2 may be prognostic factors for outcome of
therapy in nasopharyngeal cancers in Indonesia. Asian Pac
J Cancer Prev. 2010; 11:763–766.
10. Kitagawa N, Kondo S, Wakisaka N, Zen Y, Nakanishi Y,
Tsuji A, Endo K, Murono S, Yoshizaki T. Expression of
seven-in-absentia homologue 1 and hypoxia-inducible fac-
tor 1 alpha: novel prognostic factors of nasopharyngeal car-
cinoma. Cancer Lett. 2013; 331:52–57.
11. Li Z, Bian LJ, Li Y, Liang YJ, Liang HZ. Expression of
protease-activated receptor-2 (PAR-2) in patients with
nasopharyngeal carcinoma: correlation with clinicopatho-
logical features and prognosis. Pathol Res Pract. 2009;
205:542–550.
12. Mao Y, Lu MP, Lin H, Zhang da W, Liu Y, Li QD, Lv ZG,
Xu JR, Chen RJ, Zhu J. Prognostic significance of EBV
latent membrane protein 1 expression in lymphomas: evi-
dence from 15 studies. PloS one. 2013; 8:e60313.
13. Kanemitsu N, Isobe Y, Masuda A, Momose S, Higashi M,
Tamaru J, Sugimoto K, Komatsu N. Expression of Epstein-
Barr virus-encoded proteins in extranodal NK/T-cell
Lymphoma, nasal type (ENKL): differences in biologic and
clinical behaviors of LMP1-positive and -negative ENKL.
Clin Cancer Res. 2012; 18:2164–2172.
14. Claviez A, Tiemann M, Luders H, Krams M, Parwaresch R,
Schellong G, Dorffel W. Impact of latent Epstein-Barr
virus infection on outcome in children and adolescents with
Hodgkin’s lymphoma. J Clin Oncol. 2005; 23:4048–4056.
15. Dinand V, Malik A, Mohanty B, Chander B, Arya LS,
Dawar R. Low apoptotic index & bak expression in EBV-
associated childhood classical Hodgkin lymphoma. Indian
J Med Res. 2009; 130:526–532.
16. Enblad G, Sandvej K, Sundstrom C, Pallesen G, Glimelius B.
Epstein-Barr virus distribution in Hodgkin’s disease in
an unselected Swedish population. Acta Oncol. 1999;
38:425–429.
17. Sarac S, Akyol MU, Kanbur B, Poyraz A, Akyol G, Yilmaz T,
Sungur A. Bcl-2 and LMP1 expression in nasopharyngeal
carcinomas. Am J Otolaryngol. 2001; 22:377–382.
18. Song YC, Wang P. The expression of vascular endothelial
growth factor and latent membrane protein-1 and prognosis
in nasopharyngeal carcinoma. Journal of Tianjin Medical
University. 2007; 556–559.
www.impactjournals.com/oncotarget
19. Wang Y, Chen L. Expression of LMP-1, NET-1, and VEGF
in non-keratin nasopharyngeal carcinoma and its clinical
significance. Chin J Cancer Biother. 2008; 575–580.
20. Zhu H, Zeng L, Xie ZH, Hu GB, Shen LF. Study
on the Expression of LMP1, cyclinD1, and Bcl-2 in
Nasopharyngeal Carcinoma and Its Clinical Significance.
Journal of Chinese Physician. 2004; 590–592.
21. Cao W, Liu Y, Zhang H, Wang S, Zhang L, Zhang L, Sun B.
Expression of LMP- and Cyclin D protein is correlated
with an unfavorable prognosis in nasal type NK/T cell lym-
phoma. Molecular medicine reports. 2008; 1:363–368.
22. Hirose Y, Masaki Y, Sawaki T, Shimoyama K, Karasawa H,
Kawabata H, Fukushima T, Ogawa N, Wano Y, Umehara H.
Association of Epstein-Barr virus with human immuno-
deficiency virus-negative peripheral T-cell lymphomas in
Japan. Eur J Haematol. 2006; 76:109–118.
23. Ishii H, Ogino T, Berger C, Kochli-Schmitz N, Nagato T,
Takahara M, Nadal D, Harabuchi Y. Clinical usefulness of
serum EBV DNA levels of BamHI W and LMP1 for Nasal
NK/T-cell lymphoma. J Med Virol. 2007; 79:562–572.
24. Kuze T, Nakamura N, Hashimoto Y, Abe M, Wakasa H.
Clinicopathological, immunological and genetic studies of
CD30+ anaplastic large cell lymphoma of B-cell type; asso-
ciation with Epstein-Barr virus in a Japanese population. J
Pathol. 1996; 180:236–242.
25. Paydas S, Ergin M, Erdogan S, Seydaoglu G. Prognostic
significance of EBV-LMP1 and VEGF-A expres-
sions in non-Hodgkin’s lymphomas. Leuk Res. 2008;
32:1424–1430.
26. Xu G, Wang HF, He G, Xie K. Expression and significance
of p53-related proteins and LMP-1 in nasal NK/T-cell lym-
phoma. Chin J Oncol. 2009; 31:351–355.
27. Yamamoto T, Nakamura Y, Kishimoto K, Takeuchi H,
Shirakata M, Mitsuya T, Hirai K. Epstein-Barr virus (EBV)-
infected cells were frequently but dispersely detected in
T-cell lymphomas of various types by in situ hybridization
with an RNA probe specific to EBV-specific nuclear anti-
gen 1. Virus Res. 1999; 65:43–55.
28. Zhao S, Liu WP, Zhang WY, Li GD. Extranodal Nasal
Type NK/T-cell Lymphoma: the Expression of Epstein-
Barr Virus Latent Membrane Protein 1 and Its Significance
of Prognosis J Sichuan Univ. Med Sci Edi. 2005; 338–340.
29. Clarke CA, Glaser SL, Dorfman RF, Mann R, DiGiuseppe JA,
Prehn AW, Ambinder RF. Epstein-Barr virus and sur-
vival after Hodgkin disease in a population-based series of
women. Cancer. 2001; 91:1579–1587.
30. Engel M, Essop MF, Close P, Hartley P, Pallesen G,
Sinclair-Smith C. Improved prognosis of Epstein-Barr
virus associated childhood Hodgkin’s lymphoma: study of
47 South African cases. J Clin Pathol. 2000; 53:182–186.
31. Glavina-Durdov M, Jakic-Razumovic J, Capkun V, Murray P.
Assessment of the prognostic impact of the Epstein-Barr
virus-encoded latent membrane protein-1 expression in
Hodgkin’s disease. Br J Cancer. 2001; 84:1227–1234.
29322 Oncotarget
 32. Herling M, Rassidakis GZ, Medeiros LJ, Vassilakopoulos TP,
Kliche KO, Nadali G, Viviani S, Bonfante V, Giardini R,
Chilosi M, Kittas C, Gianni AM, Bonadonna G, Pizzolo G,
Pangalis GA, Cabanillas F, et al. Expression of Epstein-Barr
virus latent membrane protein-1 in Hodgkin and Reed-
Sternberg cells of classical Hodgkin’s lymphoma: associations
with presenting features, serum interleukin 10 levels, and clini-
cal outcome. Clin Cancer Res. 2003; 9:2114–2120.
33. Keresztes K, Miltenyi Z, Bessenyei B, Beck Z, Szollosi Z,
Nemes Z, Olah E, Illes A. Association between the Epstein-
Barr virus and Hodgkin’s lymphoma in the North-Eastern
part of Hungary: effects on therapy and survival. Acta
Haematol. 2006; 116:101–107.
34. Krugmann J, Tzankov A, Gschwendtner A, Fischhofer M,
Greil R, Fend F, Dirnhofer S. Longer failure-free sur-
vival interval of Epstein-Barr virus-associated classical
Hodgkin’s lymphoma: a single-institution study. Modern
pathology : an official journal of the United States and
Canadian Academy of Pathology, Inc. 2003; 16:566–573.
35. Morente MM, Piris MA, Abraira V, Acevedo A, Aguilera B,
Bellas C, Fraga M, Garcia-Del-Moral R, Gomez-Marcos F,
Menarguez J, Oliva H, Sanchez-Beato M, Montalban C.
Adverse clinical outcome in Hodgkin’s disease is associated
with loss of retinoblastoma protein expression, high Ki67
proliferation index, and absence of Epstein-Barr virus-latent
membrane protein 1 expression. Blood. 1997; 90:2429–2436.
36. Murray PG, Billingham LJ, Hassan HT, Flavell JR, Nelson PN,
Scott K, Reynolds G, Constandinou CM, Kerr DJ, Devey EC,
Crocker J, Young LS. Effect of Epstein-Barr virus infection
on response to chemotherapy and survival in Hodgkin’s dis-
ease. Blood. 1999; 94:442–447.
37. Naresh KN, Johnson J, Srinivas V, Soman CS, Saikia T,
Advani SH, Badwe RA, Dinshaw KA, Muckaden M,
Magrath I, Bhatia K. Epstein-Barr virus association in classi-
cal Hodgkin’s disease provides survival advantage to patients
and correlates with higher expression of proliferation markers
in Reed-Sternberg cells. Ann Oncol. 2000; 11:91–96.
38. Quijano S, Saavedra C, Fiorentino S, Orozco O, Bravo MM.
Epstein-Barr virus presence in Colombian Hodgkin lymphoma
cases and its relation to treatment response. Biomedica : revista
del Instituto Nacional de Salud. 2004; 24:163–173.
39. Stark GL, Wood KM, Jack F, Angus B, Proctor SJ, Taylor PR
Northern Region Lymphoma G. Hodgkin’s disease in the
elderly: a population-based study. Br J Haematol. 2002;
119:432–440.
40. Lee HS, Chang MS, Yang HK, Lee BL, Kim WH. Epstein-
barr virus-positive gastric carcinoma has a distinct protein
expression profile in comparison with epstein-barr virus-
negative carcinoma. Clin Cancer Res. 2004; 10:1698–1705.
41. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D.
Global cancer statistics. CA: a cancer journal for clinicians.
2011; 61:69–90.
42. Chen WQ, Zeng HM, Zheng RS, Zhang SW, He J. Cancer
incidence and mortality in china, 2007. Chinese journal of can-
cer research = Chung-kuo yen cheng yen chiu. 2012; 24:1–8.
www.impactjournals.com/oncotarget
43. Jarrett AF, Armstrong AA, Alexander E. Epidemiology
of EBV and Hodgkin’s lymphoma. Ann Oncol. 1996;
7:5–10.
44. Glaser SL, Lin RJ, Stewart SL, Ambinder RF, Jarrett RF,
Brousset P, Pallesen G, Gulley ML, Khan G, O’Grady J,
Hummel M, Preciado MV, Knecht H, Chan JK, Claviez A.
Epstein-Barr virus-associated Hodgkin’s disease: epide-
miologic characteristics in international data. Int J Cancer.
1997; 70:375–382.
45. Isobe Y, Aritaka N, Sasaki M, Oshimi K, Sugimoto K.
Spontaneous regression of natural killer cell lymphoma. J
Clin Pathol. 2009; 62:647–650.
46. Lee JH, Kim SH, Han SH, An JS, Lee ES, Kim YS.
Clinicopathological and molecular characteristics of
Epstein-Barr virus-associated gastric carcinoma: a meta-
analysis. J Gastroenterol Hepatol. 2009; 24:354–365.
47. Lucas KG, Salzman D, Garcia A, Sun Q. Adoptive immu-
notherapy with allogeneic Epstein-Barr virus (EBV)-specific
cytotoxic T-lymphocytes for recurrent, EBV-positive
Hodgkin disease. Cancer. 2004; 100:1892–1901.
48. Duraiswamy J, Bharadwaj M, Tellam J, Connolly G,
Cooper L, Moss D, Thomson S, Yotnda P, Khanna R.
Induction of therapeutic T-cell responses to subdominant
tumor-associated viral oncogene after immunization with
replication-incompetent polyepitope adenovirus vaccine.
Cancer Res. 2004; 64:1483–1489.
49. Mei YP, Zhou JM, Wang Y, Huang H, Deng R, Feng GK,
Zeng YX, Zhu XF. Silencing of LMP1 induces cell cycle
arrest and enhances chemosensitivity through inhibition of
AKT signaling pathway in EBV-positive nasopharyngeal
carcinoma cells. Cell cycle. 2007; 6:1379–1385.
50. Moher D, Liberati A, Tetzlaff J, Altman DG, Group P.
Preferred reporting items for systematic reviews and meta-
analyses: the PRISMA statement. Bmj. 2009; 339:b2535.
51. Parmar MK, Torri V, Stewart L. Extracting summary statis-
tics to perform meta-analyses of the published literature for
survival endpoints. Stat Med. 1998; 17:2815–2834.
52. Stang A. Critical evaluation of the Newcastle-Ottawa scale
for the assessment of the quality of nonrandomized studies
in meta-analyses. Eur J Epidemiol. 2010; 25:603–605.
53. Takahashi N, Hashizume M. A systematic review of the influ-
ence of occupational organophosphate pesticides exposure on
neurological impairment. BMJ open. 2014; 4:e004798.
54. Higgins JP, Thompson SG, Deeks JJ, Altman DG.
Measuring inconsistency in meta-analyses. Bmj. 2003;
327:557–560.
55. DerSimonian R, Laird N. Meta-analysis in clinical trials.
Control Clin Trials. 1986; 7:177–188.
56. Begg CB, Mazumdar M. Operating characteristics of a
rank correlation test for publication bias. Biometrics. 1994;
50:1088–1101.
57. Egger M, Davey Smith G, Schneider M, Minder C. Bias
in meta-analysis detected by a simple, graphical test. Bmj.
1997; 315:629–634.
29323 Oncotarget